Beta-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, which are mediators of several developmental processes. Beta-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of beta-catenin, and it is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in beta-catenin and in components regulating its stability have been found to contribute to upregulation of cell proliferation.
The xcex2-catenin protein becomes stabilized in response to Wnt/Wg, moves to the nucleus and forms complexes with the LEF1/TCF transcription factors to regulate gene expression. The level of cytosolic xcex2-catenin is determined by its interaction with a number of proteins including those in a multiprotein complex of Axin, GSK-3xcex2, APC and other proteins. The mechanism by which the Wnt signal is transmitted to this complex is unclear but it involves interaction of Wnt with its receptors, which are members of Frizzled family of seven transmembrane proteins. The stabilization of xcex2-catenin stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. This pathway is negatively regulated by Axin.
Beta-catenin is also an adherens junction protein. Adherens junctions are critical for the establishment and maintenance of epithelial layers, such as those lining organ surfaces. AJs mediate adhesion between cells, communicate a signal that neighboring cells are present, and anchor the actin cytoskeleton. In serving these roles, AJs regulate normal cell growth and behavior. At several stages of embryogenesis, wound healing, and tumor cell metastasis, cells form and leave epithelia. This process, which involves the disruption and reestablishment of epithelial cellxe2x80x94cell contacts, may be regulated by the disassembly and assembly of AJs. AJs may also function in the transmission of the xe2x80x98contact inhibitionxe2x80x99 signal, which instructs cells to stop dividing once an epithelial sheet is complete.
For many purposes, there is an interest in being able to expand stem and progenitor cells in culture. However, it is not simply a matter of maintaining cell viability for the stem cells, but also of ensuring that the stem cells increase in numbers without losing their distinctive phenotype. Current protocols for the in vitro culture of hematopoietic stem cells generally require one or a cocktail of cytokines, such as c-kit ligand (stem cell growth factor), flt-3, thrombopoietin, IL-6, etc. While a substantial increase in cell number can be obtained with such cultures, they do not provide for expanded number of cells that retain a capacity for long term repopulation of all hematopoietic lineages. See Domen and Weissman (1999) Mol Med Today 5(5):201-8; or Ziegler and Kanz (1998) Curr Opin Hematol 5(6):434-40.
Stem cells have also been grown in co-culture with stromal cells. However, it is particularly desirable to expand stem cells in a culture of known composition, rather than relying upon the presence of other cells for their maintenance.
There continues to be a strong demand for improvements in the in vitro culture of stem cells and progenitor cells. The present invention addresses this need.
Methods are provided for the expansion of progenitor or stem cells in vitro, whereby the cells retain their pluripotential phenotype after expansion. The intracellular level of xcex2-catenin is increased in the cells in culture, either by providing exogenous xcex2-catenin protein to the cell, or by introduction into the cell of a genetic construct encoding xcex2-catenin. The xcex2-catenin may be a wild type protein appropriate for the species from which the cells are derived, or preferably, a stabilized mutant form of the protein. The alteration in cellular levels of xcex2-catenin provide for increased number of cells in cycle, and leads to cultures that containing proliferating cells that maintain an undifferentiated phenotype in vitro. The expanded cell populations are useful as a source of stem cells, e.g. to reconstitute function in a host that is deficient in a particular cell lineage or lineages. In one embodiment of the invention, the target cells are hematopoietic stem cells, which may be used in transplantation to restore hematopoietic function to autologous or allogeneic recipients.